Human Lipoprotein Research Articles

Speciated Human High-Density Lipoprotein Protein Proximity Profiles

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (∼58%), phospholipids (∼21%), total cholesterol (∼16%), triglycerides (∼5%), and free cholesterol (∼4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.

Differential regulation of human apolipoprotein AI and high-density lipoprotein by fenofibrate in hapoAI and hapoAI–CIII–AIV transgenic mice

Fenofibrate, a PPAR-α agonist, lowers triglycerides (TG) and raises high-density lipoproteins (HDL-C) in humans. While fenofibrate is very effective in lowering TG, it does not raise HDL-C in humans to the same extent as seen in human apoAI transgenic (hAI-Tg) mice. We studied the mechanism of this discordance using the following compounds as tools: cholic acid that down-regulates human apoAI, and fenofibrate, that elevates hapoAI and HDL-C in hAI-Tg mice. We hypothesized that additional sequences, including apoCIII and AIV genes on chromosome 11, not present in the hapoAI transgene may be responsible for the dampened effect of fibrates on HDL-C seen in humans. For this, hAI-Tg mice with 11kb DNA segment and hapoAI–CIII–AIV-Tg mice with 33kb DNA segment harboring apoCIII and AIV genes were employed. These mice were treated with fenofibrate and cholic acid. Fenofibrate increased apoAI and HDL-C levels, and HDL size in the apoAI-Tg mice via up-regulation of the hapoAI mRNA and increased activity and mRNA of PLTP, respectively. Consistent with earlier findings, cholic acid showed similar effects of lowering HDL-C, and elevating LDL-C in hAI-Tg mice as well as in the hAI–CIII–AIV-Tg mice. Fenofibrate decreased TG and increased HDL size in hAI–CIII–AIV-Tg mice as well, but surprisingly, did not elevate serum levels of hapoAI or hepatic AI mRNA, suggesting that additional sequences not present in the hapoAI transgene (11kb) may be partly responsible for the dampened effect on HDL-C seen in hAI–CIII–AIV-Tg mice. Since hAI–CIII–AIV-Tg mouse mimics fenofibrate effects seen in humans, this transgenic mouse could serve as a better predictive model for screening HDL-C raising compounds.

Impact of serum amyloid A on high density lipoprotein composition and levels

Serum amyloid A (SAA) is an acute-phase protein mainly associated with HDL. To study the role of SAA in mediating changes in HDL composition and metabolism during inflammation, we generated mice in which the two major acute-phase SAA isoforms, SAA1.1 and SAA2.1, were deleted [SAA knockout (SAAKO) mice], and induced an acute phase to compare lipid and apolipoprotein parameters between wild-type (WT) and SAAKO mice. Our data indicate that SAA does not affect apolipoprotein A-I (apoA-I) levels or clearance under steady-state conditions. HDL and plasma triglyceride levels following lipopolysaccharide administration, as well as the decline in liver expression of apoA-I and apoA-II, did not differ between both groups of mice. The expected size increase of WT acute-phase HDL was surprisingly also seen in SAAKO acute-phase HDL despite the absence of SAA. HDLs from both mice showed increased phospholipid and unesterified cholesterol content during the acute phase. We therefore conclude that in the mouse, SAA does not impact HDL levels, apoA-I clearance, or HDL size during the acute phase and that the increased size of acute-phase HDL in mice is associated with an increased content of surface lipids, particularly phospholipids, and not surface proteins. These data need to be transferred to humans with caution due to differences in apoA-I structure and remodeling functions.

A gel-based method for purification of apolipoprotein A-I from small volumes of plasma

We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest.

Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol.

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ∼400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.

Serum opacity factor enhances HDL-mediated cholesterol efflux, esterification and anti inflammatory effects.

Serum opacity factor (SOF) is a streptococcal protein that disrupts the structure of human high density lipoproteins (HDL) releasing lipid-free apo A-I while forming a large cholesteryl ester-rich particle and a small neo HDL. Given its low cholesterol and high phospholipid contents, we tested the hypotheses that neo HDL is a better substrate for cholesterol esterification via lecithin:cholesterol acyltransferase (LCAT), better than HDL as an acceptor of THP-1 macrophage cholesterol efflux, and improves reduction of oxidized LDL-induced production of inflammatory markers. We observed that both cholesterol efflux and esterification were improved by recombinant (r)SOF treatment of whole plasma and that the underlying cause of the improved cholesterol esterification in plasma and macrophage cholesterol efflux to rSOF-treated plasma was due to the rSOF-mediated conversion of HDL to neo HDL. Moreover, the reduction of secretion of TNF-α and IL-6 by THP-1 cells by neo HDL was twice that of HDL. Studies in BHK cells overexpressing cholesterol transporters showed that efflux to neo HDL occurred primarily via ABCA1 not ABCG1. Thus, rSOF improves two steps in reverse cholesterol transport with a concomitant reduction in the release of macrophage markers of inflammation. We conclude that rSOF catalyzes a novel reaction that might be developed as a new therapy that prevents or reverses atherosclerosis via improved reverse cholesterol transport.

Human Plasma Very Low Density Lipoprotein Carries Indian Hedgehog

Hedgehog is one of the major morphogens and fulfils critical functions in both the development and maintenance of the vasculature. Hedgehog is highly hydrophobic and its diffusion toward target tissues remains only partly understood. In Drosophila, hedgehog transport via lipophorins is relevant for development, but neither the presence nor a function for a mammalian Hedgehog carried by human plasma lipoproteins has been established. We investigated the presence of Hedgehog on lipoprotein particles and determined its importance for maintaining the endothelium. LTQ-Orbitrap XL analysis of defined plasma lipoproteins revealed that Indian Hedgehog (Ihh) is present in the human very low density lipoprotein (VLDL) fraction but not in other plasma lipoprotein fractions (low density lipoprotein (LDL) and high density lipoprotein (HDL)). Using the same approach, neither Sonic Hedgehog nor Desert Hedgehog could be detected in plasma lipoprotein fractions. Most likely, primary white adipocytes are the source of Ihh loading on VLDL as both transcriptome as well as immunofluorescence analysis showed high expression of Ihh in these cells. Additionally, we show that the endothelial compartment is most likely to be affected by the presence of Ihh on VLDL. Indeed, VLDL increased survival of primary endothelial cells, suggesting that Ihh transport by VLDL is important for maintaining the human endothelium. In conclusion, our study shows that VLDL carries Ihh throughout the body in mammals and Hedgehog signaling by human plasma VLDL particles may affect blood vessel pathophysiology. A combination of three state-of-the-art technologies, proteomics, genomics, and confocal microscopy, appeared to be a powerful tool for analyzing plasma lipoprotein-associated proteins.

Proteomic Characterization of Human Plasma High Density Lipoprotein Fractionated by Gel Filtration Chromatography

Plasma levels of high density lipoprotein cholesterol (HDL-C) are inversely proportional to the incidence of cardiovascular disease. Recent applications of modern proteomic technologies have identified upward of 50 distinct proteins associated with HDL particles with many of these newly discovered proteins implicating HDL in nonlipid transport processes including complement activation, acute phase response and innate immunity. However, almost all MS-based proteomic studies on HDL to date have utilized density gradient ultracentrifugation techniques for HDL isolation prior to analysis. These involve high shear forces and salt concentrations that can disrupt HDL protein interactions and alter particle function. Here, we used high-resolution size exclusion chromatography to fractionate normal human plasma to 17 phospholipid-containing subfractions. Then, using a phospholipid binding resin, we identified proteins that associate with lipoproteins of various sizes by electrospray ionization mass spectrometry. We identified 14 new phospholipid-associated proteins that migrate with traditionally defined HDL, several of which further support roles for HDL in complement regulation and protease inhibition. The increased fractionation inherent to this method allowed us to visualize HDL protein distribution across particle size with unprecedented resolution. The observed heterogeneity across subfractions suggests the presence of HDL particle subpopulations each with distinct protein components that may prove to impart distinct physiological functions.

Measurement of lipoprotein particle sizes using dynamic light scattering

A simple method for the measurement of LDL particle sizes is needed in clinical laboratories because a predominance of small, dense LDL (sd LDL) has been associated with coronary heart disease. We applied dynamic light scattering (DLS) to measure lipoprotein particle sizes, with special reference to sd LDL. Human serum lipoproteins isolated by a combination of ultracentrifugation and gel chromatography, or by sequential ultracentrifugation, were measured for particle size using DLS. The sizes of polystyrene beads, with diameters of 21 and 28 nm according to the manufacturer, were determined by DLS as 19.3 +/- 1.0 nm (mean +/- SD, n = 11) and 25.5 +/- 1.0 nm, respectively. The coefficients of variation for the 21 and 28 nm beads were 5.1% and 3.8% (within-run, n = 11), and 2.9% and 6.2% (between-run, n = 3), respectively. The lipoprotein sizes determined by DLS for lipoprotein fractions isolated by chromatography were consistent with the elution profile. Whole serum, four isolated lipoprotein fractions (CM + VLDL + IDL, large LDL, sd LDL and HDL) and a non-lipoprotein fraction isolated by sequential ultracentrifugation were determined by DLS to be 13.1 +/- 7.5, 37.0 +/- 5.2, 21.5 +/- 0.8, 20.3 +/- 1.1, 8.6 +/- 1.5 and 8.8 +/- 2.0 nm, respectively. The proposed DLS method can differentiate the sizes of isolated lipoprotein particles, including large LDL and sd LDL, and might be used in clinical laboratories in combination with convenient lipoprotein separation.

On-Statin Cholesteryl Ester Transfer Protein Mass and Risk of Recurrent Coronary Events (from the Pravastatin or Atorvastatin Evaluation and Infection Therapy–Thrombolysis in Myocardial Infarction 22 [PROVE IT-TIMI 22] Study)

Although cholesteryl ester transfer protein (CETP) plays an important role in human lipoprotein metabolism, its relation to coronary artery disease remains controversial. The present study evaluated the relation between on-statin CETP mass and recurrent coronary events. The plasma CETP mass, measured after 4 months of statin therapy, was quantified in 3,218 patients enrolled in the Pravastatin or Atorvastatin Evaluation and Infection Therapy-Thrombolysis in Myocardial Infarction 22 (PROVE IT-TIMI 22) study. Of the 3,218 patients, 150 experienced the combined end point of recurrent myocardial infarction or death from coronary causes during a mean follow-up of 1.8 years. An increasing on-statin CETP mass was inversely related to the risk of coronary events in both unadjusted (hazard ratio [HR] per SD increase 0.77, 95% confidence interval 0.65 to 0.92, p = 0.005) and fully adjusted (HR per SD increase 0.81, 95% confidence interval 0.67 to 0.98, p = 0.027) analyses that included traditional cardiovascular risk factors. A similar trend was observed across increasing CETP mass quartiles (p trend = 0.07). A significant interaction between the CETP mass and on-treatment low-density lipoprotein (LDL) cholesterol was noted (p interaction = 0.007). A CETP mass greater than the median was associated with a decreased risk in patients with LDL cholesterol less than the median of 80 mg/dl (HR 0.52, 95% confidence interval 0.31 to 0.89, p = 0.02), but not in patients with LDL cholesterol greater than the median. In conclusion, an increasing on-statin CETP mass was inversely related to the coronary outcomes in this large clinical trial-based cohort, particularly among those with low LDL cholesterol levels. This finding is consistent with CETP facilitating reverse cholesterol transport in the setting of robust LDL clearance and might have important implications for efforts to optimally target patients with pharmacologic CETP inhibition.

An improved anion-exchange high-performance liquid chromatography method for measuring oxidized form of LDLs in human plasma

Circulating oxidized low-density lipoproteins (LDLs) (ox-LDLs) could be a sensitive marker to predict future cardiovascular events. However, a method to evaluate oxidized forms of LDLs systemically in human plasma is not yet established. In this study, we developed a novel and convenient high-performance liquid chromatography (HPLC) method for measuring ox-LDL levels in humans. Human plasma lipoproteins were separated by a modified HPLC method using a diethylaminoethyl-type anion-exchange gel column with stepwise elution. Ox-LDLs were detected by postcolumn reaction with a reagent containing cholesterol esterase and cholesterol oxidase. Particle size of each LDL fraction separated by HPLC was determined in 61 healthy subjects. Our HPLC method separated LDLs into three fractions, which were designated as LDL-1, LDL-2 and LDL-3, on the basis of their negative charges, with LDL-3 the most strongly retained fraction migrating fastest in the anodic direction, a property that reflects the net negative charge of the molecule. Western blot analysis revealed that apolipoprotein B100 in LDL-3 fraction was the most fragmented and oxidatively modified. When LDLs were oxidized in vitro by Cu2+ or 2,2-azo-bis (2-aminopropane)-2HCl or modified by various aldehydes, all of the LDL fractions migrated at the position of LDL-3. Further, among three fractions, particle size was smallest in LDL-3 fraction. Here, we developed a convenient HPLC method and identified LDL-3 as oxidized LDL fractions, although ox-LDLs were present in LDL-2 fraction, albeit lesser concentrations than in LDL-3 subfraction. Measuring ox-LDL levels in human plasma by this method may be useful to evaluate atherosclerotic disorders.

Lipoprotein profiles in SCID/uPA mice transplanted with human hepatocytes become human-like and correlate with HCV infection success

Although multiple determinants for hepatitis C virus (HCV) infection are known, it remains partly unclear what determines the human specificity of HCV infection. Presumably, the presence of appropriate entry receptors is essential, and this may explain why HCV is unable to infect nonhuman hepatocytes. However, using mice with chimeric human livers, we show in this study that the presence of human hepatocytes, and therefore human entry receptors, is not sufficient for HCV infection. In successfully transplanted SCID/Alb-uPA mice, infection with HCV is reliable only when ∼70-80% of the liver consists of human hepatocytes. We show that chimeric mice, which are hard to infect with HCV, have significant groups of human hepatocytes that are readily infected with hepatitis B virus. Thus it is unlikely that the lack of infection with HCV can simply be attributed to low hepatocyte numbers. We investigated whether the humanization of lipoprotein profiles is positively associated with infection success. We show that the lipoprotein profiles of chimeric mice become more human-like at high levels of engraftment of human hepatocytes. This and expression of markers of human lipoprotein biosynthesis, human apolipoprotein B (ApoB) and cholesterol ester transfer protein (CETP), show a strong positive correlation with successful infection. Association of HCV in the blood of chimeric mice to ApoB-containing lipoproteins is comparable to association of HCV in patient serum and provides further support for a critical role for ApoB-containing lipoproteins in the infectious cycle of HCV. Our data suggest that the weakest link in the HCV infection chain does not appear to be the presence of human hepatocytes per se. We believe that HCV infection also depends on the presence of sufficient levels of human lipoproteins.

Fatty acids increase glucose uptake and metabolism in C2C12myoblasts stably transfected with human lipoprotein lipase

Cellular effects of FFA might differ from those of lipoprotein triglyceride (TG)-derived fatty acids (TGFA). The aim of the current study was to examine the relationship between lipoprotein lipase (LPL) expression, TGFA, or FFA availability and glucose metabolism in the absence of insulin in C2C12 myoblasts. Control myoblasts or myoblasts stably transfected with human lipoprotein lipase (C2/LPL; 15-fold greater LPL activity) were incubated for 12 h in fetal bovine serum-free medium in the absence or presence of Intralipid-20. Intracellular retention of labeled medium glucose was assessed in a subset of experiments. In the presence of Intralipid, medium glucose disappearance was increased in C2/LPL cells but not in control cells. In both cell types, glucose label retention in cellular TG was increased in the presence of Intralipid; incubation with albumin-bound oleate produced similar results. In the presence of Intralipid, the LPL hydrolytic inhibitor tetrahydrolipstatin blocked excess glucose retention in cellular TG but did not significantly decrease glucose disappearance in C2/LPL cells. Changes in glucose transport or hexokinase II did not explain the altered glucose disappearance in C2/LPL cells. Our results suggest that LPL overexpression in these cells leads to chronic metabolic adaptations that alter glucose uptake and retention.

Sugar treatment of human lipoprotein particles and their separation by capillary electrophoresis

Entrapment of lipoprotein particles in the extracellular matrix of the arterial intima is a characteristic feature of the development of atherosclerosis, the disease behind myocardial infarction and stroke. In this study, sugars were exploited in the separation of lipoproteins by CE. Monosaccharides, disaccharides and one sugar alcohol used during ultracentrifugal isolation of lipoproteins prevented the strong and unfavorable adsorption of lipoprotein particles on the capillary wall, allowing their selective separation in uncoated fused silica capillary. The effect of ionic strength of the phosphate BGE solution on the separation at physiological pH was clarified. Asymmetrical flow field-flow fractionation and dynamic light scattering showed that sugars affected the structure of lipoproteins by decreasing their sizes. Although in molecular dynamics simulations, only a 19 amino acid peptide of apolipoprotein B-100 and a 15 amino acid peptide of apolipoprotein E were employed, the results also indicated a decrease in lipoprotein size, supporting the asymmetrical flow field-flow fractionation and dynamic light scattering results.

Cellular and Molecular Remodeling of the Endocytic Pathway during Differentiation of Trypanosoma brucei Bloodstream Forms

During the course of mammalian infection, African trypanosomes undergo extensive cellular differentiation, as actively dividing long slender (SL) forms progressively transform into intermediate (I) forms and finally quiescent G(1)/G(0)-locked short stumpy (ST) forms. ST forms maintain adaptations compatible with their survival in the mammalian bloodstream, such as high endocytic activity, but they already show preadaptations to the insect midgut conditions. The nutritional requirements of ST forms must differ from those of SL forms because the ST forms stop multiplying. We report that the uptake of several ligands was reduced in ST forms compared with that in SL forms. In particular, the haptoglobin-hemoglobin (Hp-Hb) complex was no longer taken up due to dramatic downregulation of its cognate receptor, TbHpHbR. As this receptor also allows uptake of trypanolytic particles from human serum, ST forms were resistant to trypanolysis by human serum lipoproteins. These observations allowed both flow cytometry analysis of SL-to-ST differentiation and the generation of homogeneous ST populations after positive selection upon exposure to trypanolytic particles. In addition, we observed that in ST forms the lysosome relocates anterior to the nucleus. Altogether, we identified novel morphological and molecular features that characterize SL-to-ST differentiation.

Lipid synthesis and secretion in HepG2 cells is not affected by ACTH

Apolipoprotein B (apoB) containing lipoproteins, i.e. VLDL, LDL and Lp(a), are consequently lowered by ACTH treatment in humans. This is also seen as reduced plasma apoB by 20-30% and total cholesterol by 30-40%, mostly accounted for by a decrease in LDL-cholesterol. Studies in hepatic cell line (HepG2) cells showed that apoB mRNA expression is reduced in response to ACTH incubation and is followed by a reduced apoB secretion, which may hypothesize that ACTH lowering apoB containing lipoproteins in humans may be mediated by the inhibition of hepatic apoB synthesis. This was recently confirmed in vivo in a human postprandial study, where ACTH reduced transient apoB48 elevation from the small intestine, however, the exogenic lipid turnover seemed unimpaired. In the present study we investigated if lipid synthesis and/or secretion in HepG2 cells were also affected by pharmacological levels of ACTH to accompany the reduced apoB output. HepG2 cells were incubated with radiolabelled precursors ([14C]acetate and [3H]glycerol) either before or during ACTH stimuli. Cellular and secreted lipids were extracted with chloroform:methanol and separated by the thin layer chromatography (TLC), and [14C]labelled cholesterol and cholesteryl ester and [3H]labelled triglycerides and phospholipids were quantitated by the liquid scintillation counting. It demonstrated that ACTH administration did not result in any significant change in neither synthesis nor secretion of the studied lipids, this regardless of presence or absence of oleic acid, which is known to stabilize apoB and enhance apoB production. The present study suggests that ACTH lowers plasma lipids in humans mainly mediated by the inhibition of apoB synthesis and did not via the reduced lipid synthesis.

Abstract 2724: Human lipoproteins, a new serum compartment for tracking biomarkers: an illustrative study of soluble VE-cadherin in malignant gliomas

Abstract BACKGROUND: Vascular endothelial (VE)-cadherin is an endothelial specific adhesion molecule of vital importance for the maintenance of endothelium integrity. Because gliomas are hypervascular tumors, the present study was designed to address the question whether Vascular endothelial-cadherin (VE-cadherin), a protein exclusively expressed in endothelial cells, could be a potential candidate biomarker for clinical use in the setting of gliomas. METHODS: Fifty-six patients newly diagnosed glioma who underwent radiochemotherapy were enrolled in this retrospective study. Serum samples were collected prior treatment. Therapeutic response to Temodal was observed by RMI in 24 patients (25 oligodendroglioma, 2 glioblastoma multiform) median age 42.15 (range 23-65) while 32 patients were resistant (5 anaplastic oligodendroglioma, 9 anaplastic astrocytomas and 17 glioblastomas multiforme), median age 51.9 (range 19-84). sVE-cadherin levels was evaluated by westernblotting and sandwich ELISA. Data were analyzed using logistic regression and receiver operating characteristics (ROC) curve analysis. RESULTS: VE-cadherin 90 kDa extracellular domain was detected in human serum by western blot and its accurate detection was dependent upon a pre-dilution in a detergent-containing buffer (Triton X-100). Of importance, we found that sVE-cadherin was carried by human lipoproteins and that the detergent allowed to separate both entities to increase protein detectability. Interestingly, we found that sVE cadherin was significantly lower in the non responders group as compared to responders. We developped an ELISA that clearly discriminate patients NR (0.669+/-0.104 AU) from patients R (0.975+/-0.098 AU (p=0.0024). Receiver-operating characteristic (ROC) curve analysis revealed that the predictive cut-off value of serum VE-cadherin for chemoresistance was 0.735 AU (area-under-the-curve 0.82; 95% confidence interval 0.71-0.87; maximal accuracy 0.875) Patients lipid profile (total cholesterol, triglycerides, LDL and HDL) were not significantly different between the two groups and could not account for the differences observed in serum VE-cadherin levels. CONCLUSIONS: We show for the first time that the lipoprotein component of serum — which is most often ignored in protein diagnostics — contains proteins that can unexpectedly serve as biomarkers for disease. This finding may open completely new avenues of biomarker research. Second, we demonstrate, one candidate marker (soluble VE-cadherin) to follow glioma therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2724.

Itraconazole, a Commonly Used Antifungal that Inhibits Hedgehog Pathway Activity and Cancer Growth

In a screen of drugs previously tested in humans we identified itraconazole, a systemic antifungal, as a potent antagonist of the Hedgehog (Hh) signaling pathway that acts by a mechanism distinct from its inhibitory effect on fungal sterol biosynthesis. Systemically administered itraconazole, like other Hh pathway antagonists, can suppress Hh pathway activity and the growth of medulloblastoma in a mouse allograft model and does so at serum levels comparable to those in patients undergoing antifungal therapy. Mechanistically, itraconazole appears to act on the essential Hh pathway component Smoothened (SMO) by a mechanism distinct from that of cyclopamine and other known SMO antagonists, and prevents the ciliary accumulation of SMO normally caused by Hh stimulation.

Genetic determinants of apolipoprotein B-100 kinetics

We review stable isotope tracer studies of apolipoprotein B-100 (apoB) kinetics concerning genetic polymorphisms and mutations that affect human lipoprotein metabolism. In obese men, the allelic combination of the apoB signal peptide, SP24, and cholesteryl ester transfer protein, CETP B1B1, is independently associated with lower VLDL apoB secretion. Microsomal triglyceride transfer protein -493G/T carriers have reduced IDL apoB and LDL apoB production as compared with controls. Mutations in cholesterol transporters (ATP-binding cassette transporter G8 and Niemann-Pick C1 Like 1) are associated with reduced VLDL apoB secretion and increased LDL apoB production and catabolism. The ATP-binding cassette transporter G8 400K variant is a significant, independent predictor of VLDL apoB secretion. Mutations in lipases (lipoprotein lipase and hepatic lipase) and transfer proteins (lecithin-cholesterol acyltransferase and cholesteryl ester transfer protein) alter their functional activity, which impact on VLDL and LDL kinetics. Mutations in genes that regulate intrahepatic apoB assembly and lipid substrate availability to the liver impact on VLDL apoB secretion. Lipoprotein tracer studies can provide functional insight into the potential impact of genetic polymorphisms in regulating apoB metabolism in humans.

Apolipoprotein M expression increases the size of nascent preβ HDL formed by ATP binding cassette transporter A1

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.